PROJECT SUMMARY A cornerstone of adaptive immune responses to infectious agents, cancers and self-tissues is the activation of CD4+ and CD8+ T cells by antigen-derived peptides (epitopes) complexed with Major Histocompatibility Complex class II and class I molecules (MHC-II and -I), respectively. Accordingly, identification of T cell-activating peptides is frequently a critical step in the development of rational prophylactic and therapeutic strategies. Most methods for identification are guided by the prevailing view that peptides are excerpted from the products of conventional transcription, RNA processing and translation. However, we and other labs have demonstrated that MHC-I- and -II-bound peptides can be derived from a wide array of non-canonical mechanisms, including initiation of translation in an alternative reading frame, initiation at a non-AUG codon, co-translational frameshifting, and proteasome-mediated post-translational peptide splicing. Currently, non-conventional epitopes are of peripheral interest, perhaps because many fundamental questions concerning their significance remain unanswered. We do not understand: 1) the fraction of all presented peptides that are non-conventional, 2) whether all the mechanisms underlying non-canonical peptide production have been described, 3) the relative frequencies with which different mechanisms produce non-conventional epitopes, 4) the extent to which non-conventional epitopes drive T cell responses to infectious agents, cancers and self-antigens, and, 5) the prevalence of MHC- II-presented non-canonical peptides, most examples thus far being MHC-I-restricted. With respect to question 4, a recent publication and our preliminary data indicate that non-canonical peptides can drive strong, immunodominant T cell responses. With respect to question 5, the Eisenlohr lab has developed a model of MHC- II-restricted peptide production that is far more complex than generally envisioned. Thus, many of the mechanisms that produce MHC-I-restricted non-conventional epitopes should apply to MHC-II. Through a highly complementary collaboration between the Eisenlohr and Ternette labs, via cutting-edge mass spectrometry, well-developed immune recognition assays, and an array of methods to identify mechanisms underlying non- conventional epitope expression, we will test the hypothesis that both MHC-I and MHC-II non-canonical epitopes, produced by a variety of mechanisms, drive a substantial portion of the TCD8+ and TCD4+ responses to influenza. In addition to potentially uncovering new fundamental cell biology, outcomes could substantially alter the view of host defenses against a high priority infectious disease, pointing to new prophylactic and therapeutic strategies. In addition, we envision the work launching several lines of future investigation, including: 1) assessing the protective capacities of non-conventional epitope-specific T cells, 2) elucidating the antigen processing machinery that produces non-conventional epitopes, 3) identifying the determinants of immunodominance and functionality with respect to conventional vs non-conventional epitopes, and 4) carrying out similar analyses with other infectious agents, cancers and autoimmune diseases.